Immobilization of Thiol-Modified Horseradish Peroxidase on Gold Nanoparticles Enhances Enzyme Stability and Prevents Proteolytic Digestion

The specificity and efficiency of enzyme-mediated reactions have the potential to positively impact many biotechnologies; however, many enzymes are easily degraded. Immobilization on a solid support has recently been explored to improve enzyme stability. This study aims to gain insights and facilitate enzyme adsorption onto gold nanoparticles (AuNPs) to form a stable bioconjugate through the installation of thiol functional groups that alter the protein chemistry. In specific, the model enzyme, horseradish peroxidase (HRP), is thiolated via Traut’s reagent to increase the robustness and enzymatic activity of the bioconjugate. This study compares HRP and its thiolated analog (THRP) to deduce the impact of thiolation and AuNP-immobilization on the enzyme activity and stability. HRP, THRP, and their corresponding bioconjugates, HRP-AuNP and THRP-AuNP, were analyzed via UV–vis spectrophotometry, circular dichroism, zeta potential, and enzyme–substrate kinetics assays. Our data show a 5-fold greater adsorption for THRP on the AuNP, in comparison to HRP, that translated to a 5-fold increase in the THRP-AuNP bioconjugate activity. The thiolated and immobilized HRP exhibited a substantial improvement in stability at elevated temperatures (50 °C) and storage times (1 month) relative to the native enzyme in solution. Moreover, HRP, THRP, and their bioconjugates were incubated with trypsin to assess the susceptibility to proteolytic digestion. Our results demonstrate that THRP-AuNP bioconjugates maintain full enzymatic activity after 18 h of incubation with trypsin, whereas free HRP, free THRP, and HRP-AuNP conjugates are rendered inactive by trypsin treatment. These results highlight the potential for protein modification and immobilization to substantially extend enzyme shelf life, resist protease digestion, and enhance biological function to realize enzyme-enabled biotechnologies.


Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of HRP and THRP
A non-reducing 4× loading buffer was prepared with glycerol (40% v/v), SDS (8%), bromophenol blue (0.4%), and 200 mM Tris pH 6.8.The loading buffer was diluted to 1× with DI water and the HRP/THRP stocks so that the HRP/THRP stocks yielded a final concentration of 1 mg/mL.After the samples rested in a boiling water bath for 5 min, 10 L was loaded into the well of an 8% polyacrylamide gel.Additionally, 5 L of Bio-Rad Precision Plus Protein Dual Color Standards was plated as the reference protein ladder.The standard procedure for an SDS-PAGE was followed per usual with the running buffer at 150 V for approximately 1 h.Afterwards, the gel was placed in a staining solution (45% MeOH, 10% acetic acid, and 0.1% Coomassie blue) over night.The next day, the gel was removed from the staining solution and placed in a destaining solution (45% MeOH and 10% acetic acid) over night.
HRP and THRP were analyzed by SDS-PAGE under non-reducing conditions to confirm that installation of the thiol did not induce unwanted protein denaturation, aggregation, or dimerization via disulfide bridging (Figure S1).HRP appears as a single protein band at ~40 kDa, as expected for the glycosylated protein.THRP presents a major band with an equivalent mobility as the HRP sample, indicating a MW of ~40 kDa.The 100 Da increase in molecular weight for each Traut's modified lysine is not expected to be resolved by the gel.A faint, secondary band is observed in the THRP sample with a molecular weight of ~80 kDa, suggesting the formation of a few dimers, likely due to intermolecular disulfide bridging.While the dimers are not desirable, they make up a small fraction of the THRP sample, and it serves as evidence for successful installation of thiols functional groups on the THRP.

Supernatant Assay to Quantify Enzyme Loading on AuNP
The protein in the supernatant is typically measured using the BCA assay.This standard approach proved inaccurate for quantitation of HRP and THRP loading, because significant amounts of enzyme adsorbed onto the walls of low-binding microcentrifuge tubes used to form the bioconjugates, ultimately leading to an overestimation of protein loading on the AuNPs.To illustrate this challenge, a 100 L aliquot of 10 g/mL HRP or THRP was added to a low-binding microcentrifuge tube for 1 h and washed three times by adding fresh buffer and vortexing.After discarding the final rinse, 1-step ABTS was added to the rinsed tube to allow for enzyme-mediated color development.Figure S2 shows the colored product formed after 30 min, establishing that both HRP and THRP adsorbed to the centrifuge tube walls, although THRP adsorbed to a greater extent.The green product is a result of enzyme-mediated oxidation of ABTS from residual enzyme adsorbed to the microcentrifuge tube walls.

[
(T)HRP]: calculated from calibration curve in Figure 4 (HRP in AuNP and THRP in AuNP linear equations) (T)HRP (g): mass adsorbed based on calculated enzyme concentration and volume of conjugate sample (106-109 L) (T)HRP (molecules): number of adsorbed molecules calculated from enzyme mass and molecular weight (44 kDa) AuNP (mL): volume of AuNP used to prepare conjugates [AuNP]: concentration of AuNP used to prepare conjugates AuNP: Number of AuNPs in conjugate calculated as volume x concentration (T)HRP/AuNP: ratio of adsorbed enzyme molecules to number of AuNPs in the conjugate suspension

Figure S3 .
Figure S3.Photograph of endpoint assay for enzyme-mediated color formation.A 100 L aliquot of buffer (left), 10 g/mL HRP (center), and 10 g/mL THRP (right) were added to microcentrifuge tubes and left at room temperature for 1 h.Samples were removed from the microcentrifuge tubes and each tube was washed three times with fresh buffer prior to the addition of 1-step ABTS.The photograph was taken 30 min after the addition of 1-step ABTS solution.

Figure S4 .
Figure S4.Endpoint assay of residual activity of HRP and THRP adsorbed to centrifuge walls.Microcentrifuge tubes filled with solutions of varying enzyme concentrations for 1 h were rinsed three times with fresh buffer, vortexing after each addition of wash buffer.Subsequently, 125 L of 1-step ABTS was added to each microcentrifuge tube and incubated 45 min to allow for color development.Next, 100 L aliquots were pipetted into wells of a 96-well microtiter plate and the absorbance at 410 nm was collected.Measured absorbance values correlate with enzyme adsorption to microcentrifuge walls and confirm loss of enzyme during the incubation step for conjugate formation.

Figure S5 .
Figure S5.Enzymatic activity of HRP and THRP conjugates prepared at different pHs.Each conjugate was prepared by incubating AuNP with 10 g/mL of enzyme for 1 h followed by purification via centrifugation prior to assessing activity.